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H B Krishnan

Publications and source records attributed to H B Krishnan.

30 records · Page 2Linked to original sources
PubMed1993

Characterization of RFRS9, a second member of the Rhizobium fredii repetitive sequence family from the nitrogen-fixing symbiont R. fredii USDA257.

The genome of the nitrogen-fixing symbiont, Rhizobium fredii USDA257, contains nine copies of repetitive sequences known as the R. fredii repetitive sequence (RFRS) family. We previously sequenced RFRS3, which is linked to symbiosis plasmid-borne nodulation genes of this organism and has substantial homology to the T-DNA of Agrobacterium rhizogenes and lesser homology to reiterated sequences of Bradyrhizobium japonicum. Here we characterize a second family member, RFRS9. The EcoRI fragment containing RFRS9 is 1,248 bp in length and contains a single 666-bp open reading frame that is flanked by perfect 8-bp inverted repeats. Nucleic and amino acid sequences corresponding to the C terminus of the putative RFRS9 protein are nearly identical to those of RFRS3, and they retain homology to DNA from A. rhizogenes. The central portion of the RFRS9 protein also appears to be related to the S locus-specific glycoprotein family of pollen stigma incompatibility glycoproteins from Brassica oleracea, which are involved in signal perception. Sequences that define the RFRS family are restricted to the open reading frame of RFRS9 and associated upstream sequences. These regions also contain a second group of repetitive sequences, which is present in four copies within the genome of USDA257. Both families of repetitive sequences are ubiquitous in R. fredii, and they are preferentially localized on symbiosis plasmids. Southern hybridization confirms that sequences homologous to RFRS9 are present in broad-host-range Rhizobium sp. strain NGR234, in A. rhizogenes, and in two biotype 3 strains of Agrobacterium tumefaciens.

Agrobacterium tumefaciens↗
PubMed1992

Differential expression of nodS accounts for the varied abilities of Rhizobium fredii USDA257 and Rhizobium sp. strain NGR234 to nodulate Leucaena spp.

Transfer of a cosmid containing nodSU from Rhizobium sp. NGR234 to Rhizobium fredii USDA257 expands the host range for nodulation to include the perennial tropical legumes, Leucaena leucocephala and Leucaena diversifolia. Complementation experiments with a series of subclones established that nodS and its associated nod-box promoter from NGR234 are sufficient to confer this extended host-range phenotype to L. leucocephala. Strain USDA257 contains its own copy of nodSU, including upstream nod-box sequences. Although both nucleotide and deduced amino acid sequences of the reading frames are homologous between the two strains, there are gaps within the promoter region and the 5'-end of nodS of USDA257. Consequently, the deduced NodS protein of USDA257 is shorter than its counterpart from NGR234, and the distance between the nod-box and the initiation codon is greater. A 36 bp deletion encompasses the extreme right border of the USDA257 nod-box and extends into the upstream leader sequence. Transcriptional fusions with both nod-boxes confirmed that the promoter from NGR234 is flavonoid-inducible, and that the nod-box from USDA257 is not. These observations were corroborated by Northern analysis with a nodS-containing Xhol fragment as hybridization probe. Flavonoid-induced cells of NGR234 gave an intense signal, but those of USDA257 yielded only a weak trace of hybridization. EcoRI fragments with homology to nodSU of USDA257 are present in 17 of 35 tested strains, including several representatives of Bradyrhizobium japonicum, Rhizobium sp., R. loti, and R. fredii. Two wild-type, leucaena-nodulating strains of Rhizobium sp. lack this homology. We conclude that a genetic defect in expression of nodS accounts for the inability of USDA257 to nodulate leucaena and that diverse rhizobia may have evolved alternative mechanisms to nodulate this legume species.

Amino Acid Sequence↗
PubMed1991

nolC, a Rhizobium fredii gene involved in cultivar-specific nodulation of soybean, shares homology with a heat-shock gene.

Rhizobium fredii strain USDA257 does not nodulate soybean (Glycine max (L.) Merr.) cultivar McCall. Mutant 257DH5, which contains a Tn5 insert in the bacterial chromosome, forms nodules on this cultivar, but acetylene-reduction activity is absent. We have sequenced the region corresponding to the site of Tn5 insertion in this mutant and find that it lies within a 1176bp open reading frame that we designate nolC. nolC encodes a protein of deduced molecular weight 43564. Nucleotide sequences homologous to nolC are present in several other Rhizobium strains, as well as Agrobacterium tumefaciens, but not in Pseudomonas syringae pathovar glycinea. nolC lacks significant sequence homology with known genes that function in nodulation, but is 61% homologous to dnaJ, an Escherichia coli gene that encodes a 41 kDa heat-shock protein. Both R. fredii USDA257 and mutant 257DH5 produce heat-shock proteins of 78, 70, 22, and 16kDa. A 4.3kb EcoRI-HindIII subclone containing nolC expresses a single 43kDa polypeptide in mini-cells. A longer, 9.4kb EcoRI fragment expresses both the 43kDa polypeptide and a 78kDa polypeptide that corresponds in size to that of the largest heat-shock protein. Thus, although nolC has strong sequence homology to dnaJ and appears to be linked to another heat-shock gene, it does not directly function in the heat-shock response.

Amino Acid Sequence↗
PubMed1989

Structure and expression of the rice glutelin multigene family.

A near full-length cDNA and three genomic clones for rice (Oryza sativa L.) glutelin were isolated and studied. Based on nucleic acid sequence and Southern blot analyses, the three isolated glutelin genomic clones were representative members of three gene subfamilies each containing five to eight copies. A comparison of DNA sequences displayed by relevant regions of these genomic clones showed that two subfamilies, represented by clones Gt1 and Gt2, were closely related and evolved by more recent gene duplication events. The 5'-flanking and coding sequences of Gt1 and Gt2 displayed at least 87% homology. In contrast, Gt3 showed little or no homology in the 5'-flanking sequences upstream of the putative CAAT boxes and exhibited significant divergence in all other portions of the gene. Conserved sequences in the 5'-flanking regions of these genes were identified and discussed in light of their potential regulatory role. The derived primary sequences of all three glutelin genomic clones showed significant homology to the legume 11 S storage proteins indicating a common gene origin. A comparison of the derived glutelin primary sequences showed that mutations were clustered in three peptide regions. One peptide region corresponded to the highly mutable hypervariable region of legume 11 S storage proteins, a potential target area for protein modification. Expression studies indicated that glutelin mRNA transcripts are differentially accumulated during endosperm development. Promoters of Gt2 and Gt3 were functional as they direct transient expression of chloramphenicol acetyltransferase in cultured plant cells.

Amino Acid Sequence↗
PubMed1987

Heat shock triggers rapid protein phosphorylation in soybean seedings.

Heat shock arrests the synthesis of many cellular proteins and simultaneously initiates expression of a unique set of proteins, termed heat shock proteins. We have found that heat shock rapidly triggers phosphorylation of a set of proteins in soybean seedlings. Although the kinetics of phosphorylation and the heat shock response are similar, the major identified phosphorylation products do not comigrate with heat shock proteins on polyacrylamide gels. Cadmium, which is known to induce the heat shock response, stimulates phosphorylation of the same set of proteins. The rapidity of phosphorylation suggests that it may play a pivotal role in sensing and transducing elevated temperature stress in plants.

Cadmium↗
PubMed1986

ADPglucose Pyrophosphorylase Is Encoded by Different mRNA Transcripts in Leaf and Endosperm of Cereals.

Western blots of soluble protein from wheat, rice, and corn showed that ADPglucose pyrophosphorylase subunits have a size of 50 kilodaltons from endosperm tissue and 43 and 46 kilodaltons from leaf. Antisera to ADPglucose pyrophosphorylase precipitated in vitro translation products of 73 and 76 kilodaltons when leaf poly(A)(+) RNA was used, whereas endosperm mRNA directed the synthesis of 50 and 56 kilodalton polypeptides. To further study the nature of these mRNA species, an ADPglucose pyrophosphorylase cDNA clone from rice endosperm polyadenylated RNA was obtained and used as a hybridization probe. Northern blots showed that ADPglucose pyrophosphorylase mRNA was slightly larger in leaf (2100 bases) than in endosperm tissue (1900 bases). These studies indicated that in cereals there are at least two tissue specific forms of ADPglucose pyrophosphorylase that are encoded by distinct mRNA transcripts. Analysis of genomic DNA by Southern blotting suggested that ADPglucose pyrophosphorylase is encoded by a small gene family.

Journal Article↗
PubMed1986

Structural Relationship among the Rice Glutelin Polypeptides.

When the glutelin protein fraction of rice (Oryza sativa L.) seeds was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, three size classes of proteins, 51 kilodaltons (kD), 34 to 37 kD, and 21 to 22 kD, as well as a contaminating prolamine polypeptide of 14 kD were detected. Antibodies were raised against these proteins and employed in studies to determine whether a precursor-product relationship existed among the glutelin components. Antibodies of the 34 to 37 kD and 21 to 22 kD polypeptides strongly reacted with the 51 kD protein, and conversely, anti-51 kD protein cross reacted with both of the putative subunits. Immunoprecipitation of in vitro translated products resulted in the synthesis of only the precursor form, indicating that the alpha and beta subunits are proteolytic products of the 51 kD precursor protein. The poly(A)(+) RNA directed in vitro translated product was about 2000 daltons larger than both the authentic glutelin precursor and the in vitro translated product from polysome run-off synthesis. Western blot analysis of the 34 to 37 kD and 21 to 22 kD polypeptides partially digested with Staphylococcus aureus V8 protease revealed distinct patterns indicating that these proteins are structurally unrelated. As observed for the glutelins, the rice prolamines are also synthesized as a precursor of 16 kD, 2000 daltons larger than the mature polypeptide. Addition of dog pancreatic microsomal membranes to a wheat germ protein translation system resulted in the processing of the prolamine preprotein but not the preproglutelin to the mature form.

Journal Article↗
PubMed1986

Gene Expression in Developing Wheat Endosperm : Accumulation of Gliadin and ADPglucose Pyrophosphorylase Messenger RNAs and Polypeptides.

The developmental accumulation pattern of messenger RNA transcripts and polypeptides for wheat gliadins and ADPglucose pyrophosphorylase was determined using cDNA and antibody probes. Gliadin mRNA was detected on Northern and RNA dot blots at 3 days after flowering, it increased 100-fold by 10 days and decreased subsequent to 14 days. The abundant mRNAs encoding alpha/beta- and gamma-type gliadins and mRNA for ADPglucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, accumulated coordinately. Despite the coordinate accumulation of their mRNA transcripts, the accumulation of gliadin and ADPglucose pyrophosphorylase polypeptides, as determined by Western blot, differed significantly. The time at which gliadin and ADPglucose pyrophosphorylase mRNAs began accumulating was also the time when the overall pattern of gene expression, as seen by two-dimensional gel electrophoresis of in vitro translation products, changed most significantly. However, the accumulation of a number of other mRNAs or polypeptides having unknown function occurred at other times during endosperm development. The pattern of expression in the earliest stages of development was strikingly similar to that of coleoptile, another rapidly growing, nonphotosynthetic tissue. Thus, the pattern of gene expression reflects the program of development observed cytologically.

Journal Article↗
PubMed1985

Wheat invertases : characterization of cell wall-bound and soluble forms.

Wheat coleoptiles have two distinct invertases, a soluble and a cell wall-bound form as indicated by results from cytochemical and biochemical studies. These enzyme activities differ in their pH optima, chromatographic behavior on diethylaminoethyl cellulose, kinetic properties, thermal stability, and response to light treatment. The soluble invertase was purified to near homogeneity by diethylaminoethyl-cellulose, concanavalin-A Sepharose, and Sephacryl S-300 chromatography. The overall purification was 175-fold with a recovery of about 26%. The holoenzyme has an apparent molecular weight of 158,000 and subunit molecular weight of 53,000 as estimated by polyacrylamide gel electrophoresis under denaturing conditions. Illumination of wheat seedlings caused an increase in the cell wall, but not the soluble, invertase activity.

Journal Article↗
PubMedDate not supplied

Sequence and analysis of the nodABC region of Rhizobium fredii USDA257, a nitrogen-fixing symbiont of soybean and other legumes.

We cloned and analyzed nodABC from Rhizobium fredii USDA257. These genes are thought to have common functions in initiation of nitrogen-fixing nodules by all rhizobia. In USDA257, they were located in a 9.2-kb EcoRI fragment that was not closely linked to either of two copies of the regulatory gene, nodD. nodABC was present in a 3,094-base pair (bp) sequenced region, which also included a consensus nod-box promoter. The three open reading frames contained 654, 642, and 1,239 bp, respectively, and encoded deduced proteins of 21.9, 23.4, and 44.7 kD. The sequence of the nodABC region of USDA257 was generally homologous with corresponding regions from other rhizobia, but it diverged significantly in the 5' non-translated region and in the 3'terminus of nodC. nodC was not translationally coupled to nodSU, as in another soybean symbiont, Bradyrhizobium japonicum, and the deduced NodC protein was the shortest of any such proteins yet described. Site-directed mutagenesis of the 9.2-kb EcoRI fragment confirmed that nodA, nodB, and nodC are essential for nodulation of soybean, but failed to identify other linked nod genes. Daidzein, a major isoflavone from soybean roots, was the most potent of nine tested flavonoids in activating a plasmid-borne nodC::lacZ fusion. The 9.2-kb fragment complemented nodA-, nodB-, and nodC- mutants of R. meliloti to the Nod+ phenotype on Medicago sativa, M. truncatula, and Trigonella foenum-graecum. Nodule numbers, percentage of nodulated plants, and shoot dry weights, however, were considerably less than in plants inoculated with mutants complemented with nodABC from R. meliloti.

Acyltransferases↗
PubMedDate not supplied

Repetitive sequences with homology to Bradyrhizobium japonicum DNA and the T-DNA of Agrobacterium rhizogenes are closely linked to nodABC of Rhizobium fredii USDA257.

We have detected strong homology between a 9.2-kb EcoRI restriction fragment from Rhizobium fredii USDA257 that contains nodABC and eight additional EcoRI fragments in DNA digests from this organism. A series of repetitive sequences responsible for this hybridization lies within a 0.95-kb HindIII/SalI subfragment about 1-kb upstream of nodA. This subfragment also hybridizes to multiple restriction fragments from nine other strains of R. fredii, but only one is common to all strains. The 0.95-kb subfragment does not hybridize to genomic DNA from 17 other strains of fast-growing rhizobia, but there is weak homology to two fragments from Rhizobium sp. strain NGR234. We sequenced 2,432 base pairs (bp) of the region encompassing the repetitive sequences. It contains 65 separate 8- to 11-bp inverted and direct repeats, as well as two large open reading frames (ORFs) that overlap on opposite strands. ORF1 reads in the same direction as nodABC, contains 1,071 bp, and encodes a 40.6-kD protein. It has 74% sequence homology to an ORF within the T-DNA of Agrobacterium rhizogenes and similar homology to a series of repetitive sequences from Bradyrhizobium japonicum. ORF2 (981 bp) reads in the opposite direction, encodes a 34.7-kD protein, and has partial identity with a second ORF from A. rhizogenes. We could detect no poly(A)+ nodule transcripts with homology to ORF1 and ORF2. The eight sets of repetitive sequences found in other EcoRI fragments of the genome were cloned from USDA257 on separate cosmids. Some of these cosmids appear to overlap, and two have fragments with homology to nifKDH.

Amino Acid Sequence↗